Yersinia pestis, the etiologic agent of plague, uses a type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. The assembly and function of the Yersinia T3S apparatus is dependent upon 21 essential Yersinia secretion (Ysc) proteins. Following assembly of a functional base structure, the T3S apparatus secretes only early substrates (YscF, YscI and YscP) that function to assemble the rod (YscI) and needle (YscF) structures; however, little is known about the unique features of YscF and other early substrates that enable their selective recognition and secretion. In this proposal, we will characterize the unique molecular features that identify YscF as an early T3S substrate; as well as the components of the T3S apparatus that recognize these features. Importantly, cytoplasmic YscF is found exclusively in a 1:1:1 complex with its heterodimeric YscE/YscG chaperone, an interaction that is essential to prevent proteolytic degradation of YscF. We also provide experimental evidence that the YscE/YscG chaperone is essential for YscF secretion independent of its role in stabilizing YscF. Initially, we will investigate the role of the YscF N-terminal secretion signal in the hierarchical secretion of YscF. In addition, YscE and YscG point mutants that bind and stabilize YscF, but do not secrete YscF will be identified. Together, these studies will define the unique molecular features of YscF and the YscE/YscG chaperone that are required for recognition of the YscEFG complex and selective secretion of YscF. Finally, we will use characterize the role of a YscKYscQYscL sorting platform and YscN ATPase in the recognition of native and mutant YscEFG complexes. These studies will define the recognition events that allow the T3S apparatus to differentiate early and late T3S substrates, a process that is essential for establishing a secretion hierarchy.